In vitro cultivation of adult human chondrocytes : importance of culture system, oxygen and zonal differences

Articular cartilage exhibits limited intrinsic regenerative capacity and focal tissue defects can lead to the development of osteoarthritis (OA), a painful and debilitating loss of cartilage tissue. In Australia, 1.4 million people are affected by OA and its prevalence is increasing in line with current demographics. As treatment options are limited, new therapeutic approaches are being investigated including biological resurfacing of joints with tissue-engineered cartilage. Despite some progress in the field, major challenges remain to be addressed for large scale clinical success. For example, large numbers of chondrogenic cells are required for cartilage formation, but chondrocytes lose their chondrogenic phenotype (dedifferentiate) during in vitro propagation. Additionally, the zonal organization of articular cartilage is critical for normal cartilage function, but development of zonal structure has been largely neglected in cartilage repair strategies. Therefore, we hypothesised that culture conditions for freshly isolated human articular chondrocytes from non-OA and OA sources can be improved by employing microcarrier cultures and a reduced oxygen environment and that oxygen is a critical factor in the maintenance of the zonal chondrocyte phenotype. Microcarriers have successfully been used to cultivate bovine chondrocytes, and offer a potential alternative for clinical expansion of human chondrocytes. We hypothesised that improved yields can be achieved by propagating human chondrocytes on microcarriers. We found that cells on microcarriers acquired a flattened, polygonal morphology and initially proliferated faster than monolayercultivated cells. However, microcarrier cultivation over four weeks did not improve growth rates or the chondrogenic potential of non-OA and OA human articular chondrocytes over conventional monolayer cultivation. Based on these observations, we aimed to optimise culture conditions by modifying oxygen tension, to more closely reflect the in vivo environment. We found that propagation at 5% oxygen tension (moderate hypoxia) did not improve proliferation or redifferentiation capacity of human osteoarthritic chondrocytes. Moderate hypoxia increased the expression of chondrogenic markers during redifferentiation. However, osteoarthritic chondrocytes cultivated on microcarriers exhibited lower expression levels of chondrogenic surface marker proteins and had at best equivalent redifferentiation capacities compared to monolayer-cultured cells. This suggests that monolayer culture with multiple passaging potentially selects for a subpopulation of cells with higher differentiation capacity, which are otherwise rare in osteoarthritic, aged cartilage. However, fibroblastic proteins were found to be highly expressed in all cultures of human osteoarthritic chondrocytes indicating the presence of a high proportion of dedifferentiated, senescent cells with a chondrocytic phenotype that was not rescued by moderate hypoxia. The different zones of cartilage support chondrocyte subpopulations, which exhibit characteristic protein expression and experience varying oxygen tensions. We, therefore, hypothesised that oxygen tension affects the zonal marker expression of human articular chondrocytes isolated from the different cartilage layers. We found that zonal chondrocytes maintained these phenotypic differences during in vitro cultivation. Low oxygen environments favoured the expression of the zonal marker proteoglycan 4 in superficial cells, most likely through the promotion of chondrogenesis. The putative zonal markers clusterin and cartilage intermediate layer protein were found to be expressed by all subpopulations of human osteoarthritic chondrocytes ex vivo and, thus, may not be reliable predictors of in vitro stratification using these clinically relevant cells. The findings in this thesis underline the importance of considering low oxygen conditions and zonal stratification when creating native-like cartilaginous constructs. We have not yet found the right cues to successfully cultivate clinically-relevant human osteoarthritic chondrocytes in vitro. A more thorough understanding of chondrocyte biology and the processes of chondrogenesis are required to ensure the clinical success of cartilage tissue engineering.

Design and deployment of a remote robust sensor network : experiences from an outdoor water quality monitoring network

This paper investigates a wireless sensor network deployment - monitoring water quality, e.g. salinity and the level of the underground water table - in a remote tropical area of northern Australia. Our goal is to collect real time water quality measurements together with the amount of water being pumped out in the area, and investigate the impacts of current irrigation practice on the environments, in particular underground water salination. This is a challenging task featuring wide geographic area coverage (mean transmission range between nodes is more than 800 meters), highly variable radio propagations, high end-to-end packet delivery rate requirements, and hostile deployment environments. We have designed, implemented and deployed a sensor network system, which has been collecting water quality and flow measurements, e.g., water flow rate and water flow ticks for over one month. The preliminary results show that sensor networks are a promising solution to deploying a sustainable irrigation system, e.g., maximizing the amount of water pumped out from an area with minimum impact on water quality.

The Green Falcon - Solar Powered Unmanned Aerial Vehicle

In recent months the extremes of Australia’s weather have affected, killed a good number of people and millions of dollars lost. Contrary to a manned aircraft or a helicopter; which have restricted air time, a UAS or a group of UAS could provide 24 hours coverage of the disaster area and be instrumented with infrared cameras to locate distressed people and relay information to emergency services. The solar powered UAV is capable of carrying a 0.25Kg payload consuming 0.5 watt and fly continuously for at low altitude for 24 hrs ,collect the data and create a special distribution . This system, named Green Falcon, is fully autonomous in navigation and power generation, equipped with solar cells covering its wing, it retrieves energy from the sun in order to supply power to the propulsion system and the control electronics, and charge the battery with the surplus of energy. During the night, the only energy available comes from the battery, which discharges slowly until the next morning when a new cycle starts. The prototype airplane was exhibited at the Melbourne Museum form Nov09 to Feb 2010.

Top Gear, top journalism : three lessons for political journalists from the world's most popular TV show

This article examines the BBC program Top Gear, discussing why it has become one of the world’s most-watched TV programs, and how it has very successfully captivated an audience who might otherwise not be particularly interested in cars. The analysis of the show is here framed in the form of three ‘lessons’ for journalists, suggesting that some of the entertaining (and highly engaging) ways in which Top Gear presents information to its viewers could be usefully applied in the coverage of politics – a domain of knowledge which, like cars, many citizens find abstract or boring.

Visual control of robots : high-performance visual servoing

The application of high-speed machine vision for close-loop position control, or visual servoing, of a robot manipulator. It provides a comprehensive coverage of all aspects of the visual servoing problem: robotics, vision, control, technology and implementation issues. While much of the discussion is quite general the experimental work described is based on the use of a high-speed binary vision system with a monocular "eye-in-hand" camera.

Developing regional precise positioning services using the legacy and future GNSS receivers

This paper presents an overview of technical solutions for regional area precise GNSS positioning services such as in Queensland. The research focuses on the technical and business issues that currently constrain GPS-based local area Real Time Kinematic (RTK) precise positioning services so as to operate in future across larger regional areas, and therefore support services in agriculture, mining, utilities, surveying, construction, and others. The paper first outlines an overall technical framework that has been proposed to transition the current RTK services to future larger scale coverage. The framework enables mixed use of different reference GNSS receiver types, dual- or triple-frequency, single or multiple systems, to provide RTK correction services to users equipped with any type of GNSS receivers. Next, data processing algorithms appropriate for triple-frequency GNSS signals are reviewed and some key performance benefits of using triple carrier signals for reliable RTK positioning over long distances are demonstrated. A server-based RTK software platform is being developed to allow for user positioning computations at server nodes instead of on the user's device. An optimal deployment scheme for reference stations across a larger-scale network has been suggested, given restrictions such as inter-station distances, candidates for reference locations, and operational modes. For instance, inter-station distances between triple-frequency receivers can be extended to 150km, which doubles the distance between dual-frequency receivers in the existing RTK network designs.

An examination of communication across cultures in news media and at informal/personal levels : with concentration on relations among two South East Asian countries and Australia and those two countries and Germany

In the age of globalisation dominated by mass communication, the flow of information contributes to a big extent to the worldviews of its "global citizens". From this point of view the mass media can be seen as one of the most salient sources of cross-cultural communication. This study investigates mass communication across cultures, focusing on South East Asia (Malaysia and Singapore), Australia and Germany. The centre of attention is the Western media coverage of South East Asia and vice versa. In this context a content analysis of newspapers of the three regions has been conducted. In addition, working practices and conditions of Western foreign correspondents in South East Asia have been examined. Apart from the investigation of inter-cultural media coverage, another focus of attention will be the examination of two levels of communication: The business level, concentrating on issues like e.g. the Asian business etiquette; and the private level, looking into the transition to a different culture from the perspective of Australian and German expatriates. Apart from investigating mass communication across cultures and to provide a written analysis of the findings, a series of radio documentaries in English and in German has been produced. They cover the following issues: Foreign correspondents in South East Asia, the expatriate-lifestyle of Australians and Germans in South East Asia, business etiquette in Asia, student exchange Germany-Asia, image and prejudices East-West and Tourism.

Safety-restraint use rate as function of law enforcement and other factors : preliminary analysis

Persistent use of safety restraints prevents deaths and reduces the severity and number of injuries resulting from motor vehicle crashes. However, safety-restraint use rates in the United States have been below those of other nations with safety-restraint enforcement laws. With a better understanding of the relationship between safety-restraint law enforcement and safety-restraint use, programs can be implemented to decrease the number of deaths and injuries resulting from motor vehicle crashes. Does safety-restraint use increase as enforcement increases? Do motorists increase their safety-restraint use in response to the general presence of law enforcement or to targeted law enforcement efforts? Does a relationship between enforcement and restraint use exist at the countywide level? A logistic regression model was estimated by using county-level safety-restraint use data and traffic citation statistics collected in 13 counties within the state of Florida in 1997. The model results suggest that safety-restraint use is positively correlated with enforcement intensity, is negatively correlated with safety-restraint enforcement coverage (in lanemiles of enforcement coverage), and is greater in urban than rural areas. The quantification of these relationships may assist Florida and other law enforcement agencies in raising safety-restraint use rates by allocating limited funds more efficiently either by allocating additional time for enforcement activities of the existing force or by increasing enforcement staff. In addition, the research supports a commonsense notion that enforcement activities do result in behavioral response.

Multiple camera management using wide baseline matching

Camera calibration information is required in order for multiple camera networks to deliver more than the sum of many single camera systems. Methods exist for manually calibrating cameras with high accuracy. Manually calibrating networks with many cameras is, however, time consuming, expensive and impractical for networks that undergo frequent change. For this reason, automatic calibration techniques have been vigorously researched in recent years. Fully automatic calibration methods depend on the ability to automatically find point correspondences between overlapping views. In typical camera networks, cameras are placed far apart to maximise coverage. This is referred to as a wide base-line scenario. Finding sufficient correspondences for camera calibration in wide base-line scenarios presents a significant challenge. This thesis focuses on developing more effective and efficient techniques for finding correspondences in uncalibrated, wide baseline, multiple-camera scenarios. The project consists of two major areas of work. The first is the development of more effective and efficient view covariant local feature extractors. The second area involves finding methods to extract scene information using the information contained in a limited set of matched affine features. Several novel affine adaptation techniques for salient features have been developed. A method is presented for efficiently computing the discrete scale space primal sketch of local image features. A scale selection method was implemented that makes use of the primal sketch. The primal sketch-based scale selection method has several advantages over the existing methods. It allows greater freedom in how the scale space is sampled, enables more accurate scale selection, is more effective at combining different functions for spatial position and scale selection, and leads to greater computational efficiency. Existing affine adaptation methods make use of the second moment matrix to estimate the local affine shape of local image features. In this thesis, it is shown that the Hessian matrix can be used in a similar way to estimate local feature shape. The Hessian matrix is effective for estimating the shape of blob-like structures, but is less effective for corner structures. It is simpler to compute than the second moment matrix, leading to a significant reduction in computational cost. A wide baseline dense correspondence extraction system, called WiDense, is presented in this thesis. It allows the extraction of large numbers of additional accurate correspondences, given only a few initial putative correspondences. It consists of the following algorithms: An affine region alignment algorithm that ensures accurate alignment between matched features; A method for extracting more matches in the vicinity of a matched pair of affine features, using the alignment information contained in the match; An algorithm for extracting large numbers of highly accurate point correspondences from an aligned pair of feature regions. Experiments show that the correspondences generated by the WiDense system improves the success rate of computing the epipolar geometry of very widely separated views. This new method is successful in many cases where the features produced by the best wide baseline matching algorithms are insufficient for computing the scene geometry.

An investigation of dose calculation accuracy in intensity-modulated radiotherapy of sites in the head & neck

Knowledge of the accuracy of dose calculations in intensity-modulated radiotherapy of the head and neck is essential for clinical confidence in these highly conformal treatments. High dose gradients are frequently placed very close to critical structures, such as the spinal cord, and good coverage of complex shaped nodal target volumes is important for long term-local control. A phantom study is presented comparing the performance of standard clinical pencil-beam and collapsed-cone dose algorithms to Monte Carlo calculation and three-dimensional gel dosimetry measurement. All calculations and measurements are normalized to the median dose in the primary planning target volume, making this a purely relative study. The phantom simulates tissue, air and bone for a typical neck section and is treated using an inverse-planned 5-field IMRT treatment, similar in character to clinically used class solutions. Results indicate that the pencil-beam algorithm fails to correctly model the relative dose distribution surrounding the air cavity, leading to an overestimate of the target coverage. The collapsed-cone and Monte Carlo results are very similar, indicating that the clinical collapsed-cone algorithm is perfectly sufficient for routine clinical use. The gel measurement shows generally good agreement with the collapsed-cone and Monte Carlo calculated dose, particularly in the spinal cord dose and nodal target coverage, thus giving greater confidence in the use of this class solution.

Mineralization capacity of Runx2/Cbfa1-genetically engineered fibroblasts is scaffold dependent

Development of tissue-engineered constructs for skeletal regeneration of large critical-sized defects requires the identification of a sustained mineralizing cell source and careful optimization of scaffold architecture and surface properties. We have recently reported that Runx2-genetically engineered primary dermal fibroblasts express a mineralizing phenotype in monolayer culture, highlighting their potential as an autologous osteoblastic cell source which can be easily obtained in large quantities. The objective of the present study was to evaluate the osteogenic potential of Runx2-expressing fibroblasts when cultured in vitro on three commercially available scaffolds with divergent properties: fused deposition-modeled polycaprolactone (PCL), gas-foamed polylactide-co-glycolide (PLGA), and fibrous collagen disks. We demonstrate that the mineralization capacity of Runx2-engineered fibroblasts is scaffold dependent, with collagen foams exhibiting ten-fold higher mineral volume compared to PCL and PLGA matrices. Constructs were differentially colonized by genetically modified fibroblasts, but scaffold-directed changes in DNA content did not correlate with trends in mineral deposition. Sustained expression of Runx2 upregulated osteoblastic gene expression relative to unmodified control cells, and the magnitude of this expression was modulated by scaffold properties. Histological analyses revealed that matrix mineralization co-localized with cellular distribution, which was confined to the periphery of fibrous collagen and PLGA sponges and around the circumference of PCL microfilaments. Finally, FTIR spectroscopy verified that mineral deposits within all Runx2-engineered scaffolds displayed the chemical signature characteristic of carbonate-containing, poorly crystalline hydroxyapatite. These results highlight the important effect of scaffold properties on the capacity of Runx2-expressing primary dermal fibroblasts to differentiate into a mineralizing osteoblastic phenotype for bone tissue engineering applications.

Osteogenic induction of human bone marrow-derived mesenchymal progenitor cells in novel synthetic polymer-hydrogel matrices

The aim of this project was to investigate the in vitro osteogenic potential of human mesenchymal progenitor cells in novel matrix architectures built by means of a three-dimensional bioresorbable synthetic framework in combination with a hydrogel. Human mesenchymal progenitor cells (hMPCs) were isolated from a human bone marrow aspirate by gradient centrifugation. Before in vitro engineering of scaffold-hMPC constructs, the adipogenic and osteogenic differentiation potential was demonstrated by staining of neutral lipids and induction of bone-specific proteins, respectively. After expansion in monolayer cultures, the cells were enzymatically detached and then seeded in combination with a hydrogel into polycaprolactone (PCL) and polycaprolactone-hydroxyapatite (PCL-HA) frameworks. This scaffold design concept is characterized by novel matrix architecture, good mechanical properties, and slow degradation kinetics of the framework and a biomimetic milieu for cell delivery and proliferation. To induce osteogenic differentiation, the specimens were cultured in an osteogenic cell culture medium and were maintained in vitro for 6 weeks. Cellular distribution and viability within three-dimensional hMPC bone grafts were documented by scanning electron microscopy, cell metabolism assays, and confocal laser microscopy. Secretion of the osteogenic marker molecules type I procollagen and osteocalcin was analyzed by semiquantitative immunocytochemistry assays. Alkaline phosphatase activity was visualized by p-nitrophenyl phosphate substrate reaction. During osteogenic stimulation, hMPCs proliferated toward and onto the PCL and PCL-HA scaffold surfaces and metabolic activity increased, reaching a plateau by day 15. The temporal pattern of bone-related marker molecules produced by in vitro tissue-engineered scaffold-cell constructs revealed that hMPCs differentiated better within the biomimetic matrix architecture along the osteogenic lineage.